The role of cis-regulatory elements in the inheritance of transcriptional memory through mitosis.

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PROJECT SUMMARY The goal of this proposal is to determine how cell identity, as defined by a transcriptional program, is inherited through mitosis by systematically evaluating the role of specific cis-regulatory elements and trans- acting factors. The central hypothesis is that cis-regulatory elements at gene promoters maintain transcriptional competency through mitosis while trans interactions with enhancers mediate the timely and appropriate level of gene expression during mitotic exit. This proposal will leverage how the underlying DNA sequence determines the multifaceted interactions at promoters and cell-type specific enhancers that ensure cells faithfully re- establish proper transcriptional programs. This will be evaluated in cells with a stable identity and expanded to understand how this is mediated in the context of early development, when cells undergo constant fate decisions. While it was previously thought that chromatin condensation during mitosis excluded most proteins and thereby transcription-related processes, recent studies originally emanating from the Zaret lab now indicate that transcription is active at a low level and promoters, but not most enhancers, are accessible and maintain active histone modifications [1-7]; and mitotic chromatin arms retain areas of dynamic chromatin [8]. Although genome-wide measurements of histone modifications, chromatin accessibility and chromatin organization suggest mitotic changes at various cis-regulatory elements, they are unable to garner high- resolution mechanistic insight into how mitotic memory is retained at promoters and acted upon, during mitotic exit, by enhancers. While components involved in transcription such as the TATA-binding protein TBP, the elongating form of RNAP2, and transcription factors (TFs) such as GATA1, FOXA1, and ESRRB are detected on mitotic chromatin; many of these TFs do not remain bound to their interphase enhancer targets, thus how these factors function at specific loci to transmit transcription memory through mitosis remains elusive [5-7, 9- 13]. Therefore, this proposal aims to determine how cell fate is preserved through cell division by first evaluating the functional requirement for promoters in mitotic transcription, and then the mechanisms employed by liver-specific transcription factors to mediate enhancer activation upon mitotic exit. This will be assessed in the liver-derived HUH7 human cell line, which is highly amenable to mitotic synchronization methods, and expanded to determine the role of identified factors in pluripotent and endoderm-differentiated human embryonic stem cells. This proposal will expand our understanding of the molecular mechanisms that maintain cell identity and cell fate specification, thereby improving our ability to target and modulate stem cells for therapeutic value.